Distinct Internalization Pathways of Human Amylin Monomers and Its Cytotoxic Oligomers in Pancreatic Cells

Saurabh Trikha,Aleksandar M. Jeremic,Jie Zheng

doi:10.1371/journal.pone.0073080

Abstract

Toxic human amylin oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (TTDM). Although recent studies have shown that pancreatic cells can recycle amylin monomers and toxic oligomers, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Using pancreatic rat insulinoma (RIN-m5F) beta (β)-cells and human islets as model systems we show that monomers and oligomers cross the plasma membrane (PM) through both endocytotic and non-endocytotic (translocation) mechanisms, the predominance of which is dependent on amylin concentrations and incubation times. At low (≤100 nM) concentrations, internalization of amylin monomers in pancreatic cells is completely blocked by the selective amylin-receptor (AM-R) antagonist, AC-187, indicating an AM-R dependent mechanism. In contrast at cytotoxic (µM) concentrations monomers initially (1 hour) enter pancreatic cells by two distinct mechanisms: translocation and macropinocytosis. However, during the late stage (24 hours) monomers internalize by a clathrin-dependent but AM-R and macropinocytotic independent pathway. Like monomers a small fraction of the oligomers initially enter cells by a non-endocytotic mechanism. In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors. This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells. Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin’s molecular forms, thereby serving a cyto-protective role in these cells.

Highlights

Human islet amyloid polypeptide or amylin is a 37- amino acid peptide hormone produced and co-secreted with insulin by pancreatic beta-cells [1,2,3,4]
We investigated the roles of amylin receptor (AMR) and endocytosis in the uptake and toxicity of human amylin in cultured pancreatic RIN-m5F and human islet cells
As human amylin is non-toxic at low concentrations and cytotoxic at higher concentrations [50], we examined the mechanism of amylin monomer and oligomer internalization at these two distinct concentrations, aiming to understand how cells deal with amylin overload

Summary

Introduction

Human islet amyloid polypeptide or amylin is a 37- amino acid peptide hormone produced and co-secreted with insulin by pancreatic beta-cells [1,2,3,4]. Other studies show that amylin aggregation may contribute to the pathogenesis of TTDM [28,29,30,31,32] Both intracellular and extracellular accumulations of human amylin oligomers and aggregates in the pancreas are reported to be cytotoxic to islet b-cells, the loss of which correlates with progression of TTDM [26,27,28,32,33]. Direct contact of these oligomers and aggregates with the b-cell PM is required to elicit apoptosis [11,30,34]. Endoplasmic reticulum stress response [39], activations of stress-activated kinases [40], induction of reactive oxidative stress species or radicals [41,42] and Ca2+ overload [35,43] are other possible mechanisms of amylin-induced toxicity in cells

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Conclusion